Background and Aims One of the prerequisites for polyploid research in natural systems is knowledge of the geographical distribution of cytotypes. Here inter- and intrapopulational ploidy diversity was examined in the Gymnadenia conopsea aggregate in central Europe and potential explanations and evolutionary consequences of the observed spatial patterns investigated. Methods DAPI flow cytometry supplemented by confirmatory chromosome counts was used to determine ploidy in 3581 samples of the G. conopsea aggregate from 43 populations. The fine-scale spatial pattern of cytotype distribution (intra-and interploidy associations) was analysed with univariate and bivariate K-functions. Key Results Gymnadenia tissues undergo a progressively partial endoreplication, which accounts for about 60% and 75% of the total genome in G. conopsea and G. densiflora, respectively. Flow cytometric profiles are therefore species-specific and can be used as a marker for rapid and reliable species recognition. Two majority (4x, 8x) and three minority (6x, 10x, 12x) cytotypes were found, often in mixed-ploidy populations (harbouring up to all five different ploidy levels). The scarcity of the minority cytotypes (about 2.7%) suggests the existence of strong pre- or postzygotic mating barriers. Spatial structure was observed in plots of populations with the highest cytotype variation, including clumping of individuals of the same ploidy and negative association between tetra-and octoploids. Conclusions The remarkable ploidy coexistence in the G. conopsea aggregate has reshaped our perception of intrapopulational ploidy diversity under natural conditions. This system offers unique opportunities for studying processes governing the formation and establishment of polyploids and assessing the evolutionary significance of the various pre- and postzygotic mating barriers that maintain this ploidy mixture.